Mef2 interacts with the Notch pathway during adult muscle development in Drosophila melanogaster.

Myogenesis of indirect flight muscles (IFMs) in Drosophila melanogaster follows a well-defined cellular developmental scheme. During embryogenesis, a set of cells, the Adult Muscle Precursors (AMPs), are specified. These cells will become proliferating myoblasts during the larval stages which will then give rise to the adult IFMs. Although the cellular aspect of this developmental process is well studied, the molecular biology behind the different stages is still under investigation. In particular, the interactions required during the transition from proliferating myoblasts to differentiated myoblasts ready to fuse to the muscle fiber. It has been previously shown that the Notch pathway is active in proliferating myoblasts, and that this pathway is inhibited in developing muscle fibers. Furthermore, the Myocyte Enhancing Factor 2 (Mef2), Vestigial (Vg) and Scalloped (Sd) transcription factors are necessary for IFM development and that Vg is required for Notch pathway repression in differentiating fibers. Here we examine the interactions between Notch and Mef2 and mechanisms by which the Notch pathway is inhibited during differentiation. We show that Mef2 is capable of inhibiting the Notch pathway in non myogenic cells. A previous screen for Mef2 potential targets identified Delta a component of the Notch pathway. Dl is expressed in Mef2 and Sd-positive developing fibers. Our results show that Mef2 and possibly Sd regulate a Dl enhancer specifically expressed in the developing IFMs and that Mef2 is required for Dl expression in developing IFMs.

The insulin receptor is required for the development of the Drosophila peripheral nervous system.

The Insulin Receptor (InR) in Drosophila presents features conserved in its mammalian counterparts. InR is required for growth; it is expressed in the central and embryonic nervous system and modulates the time of differentiation of the eye photoreceptor without altering cell fate. We show that the InR is required for the formation of the peripheral nervous system during larval development and more particularly for the formation of sensory organ precursors (SOPs) on the fly notum and scutellum. SOPs arise in the proneural cluster that expresses high levels of the proneural proteins Achaete (Ac) and Scute (Sc). The other cells will become epidermis due to lateral inhibition induced by the Notch (N) receptor signal that prevents its neighbors from adopting a neural fate. In addition, misexpression of the InR or of other components of the pathway (PTEN, Akt, FOXO) induces the development of an abnormal number of macrochaetes that are Drosophila mechanoreceptors. Our data suggest that InR regulates the neural genes ac, sc and sens. The FOXO transcription factor which is localized in the cytoplasm upon insulin uptake, displays strong genetic interaction with the InR and is involved in Ac regulation. The genetic interactions between the epidermal growth factor receptor (EGFR), Ras and InR/FOXO suggest that these proteins cooperate to induce neural gene expression. Moreover, InR/FOXO is probably involved in the lateral inhibition process, since genetic interactions with N are highly significant. These results show that the InR can alter cell fate, independently of its function in cell growth and proliferation.

Interaction with the Yes-associated protein (YAP) allows TEAD1 to positively regulate NAIP expression.

Although the expression of the neuronal apoptosis inhibitory protein (NAIP) gene is considered involved in apoptosis suppression as well as in inflammatory response, the molecular basis of the NAIP gene expression is poorly understood. Here we show that the TEA domain protein 1 (TEAD1) is able to positively activate the transcription of NAIP. We further demonstrate that this regulation is mediated by the presence of the endogenous Yes associated protein (YAP) cofactor, and requires the interaction with YAP. We finally identified an intronic region of the NAIP gene responding to TEAD1/YAP activity, suggesting that regulation of NAIP by TEAD1/YAP is at the transcriptional level.

The Hippo kinase promotes Scalloped cytoplasmic localization independently of Warts in a CRM1/Exportin1-dependent manner in Drosophila.

Scalloped (SD) is a transcription factor characterized by a TEA/ATTS DNA binding domain. To activate transcription, SD must interact with its coactivators, including Yorkie (YKI) or Vestigial (VG). YKI is the downstream effector of the Hippo signaling pathway that plays a key role in the control of tissue growth. The core components of this pathway are two kinases, Hippo (HPO) and Warts (WTS), which negatively regulate the activity of the SD/YKI complex, retaining YKI in the cytoplasm. We previously showed that HPO kinase can also reduce SD/VG transcriptional activity in Drosophila S2 cells. We further investigated the relationship between the SD/VG complex and the Hippo pathway. We show here that HPO overexpression suppresses overgrowth induced by SD/VG in vivo during Drosophila development. Using S2 cells, we show that HPO promotes the translocation of SD to the cytoplasm in a CRM1-dependent manner, thereby inhibiting the induction of SD/VG target genes. Using RNAi-mediated depletion of yki and a mutant SD protein unable to interact with YKI, we demonstrate that HPO regulates SD localization independently of YKI. This function requires HPO kinase activity, yet surprisingly, not its downstream effector kinase WTS. Taken together, these observations reveal a new and unexpected role of HPO kinase in the regulation of a transcription factor independently of YKI.

Alteration of TEAD1 expression levels confers apoptotic resistance through the transcriptional up-regulation of Livin.

BACKGROUND: TEA domain (TEAD) proteins are highly conserved transcription factors involved in embryonic development and differentiation of various tissues. More recently, emerging evidences for a contribution of these proteins towards apoptosis and cell proliferation regulation have also been proposed. These effects appear to be mediated by the interaction between TEAD and its co-activator Yes-Associated Protein (YAP), the downstream effector of the Hippo tumour suppressor pathway. METHODOLOGY/PRINCIPAL FINDINGS: We further investigated the mechanisms underlying TEAD-mediated apoptosis regulation and showed that overexpression or RNAi-mediated silencing of the TEAD1 protein is sufficient to protect mammalian cell lines from induced apoptosis, suggesting a proapoptotic function for TEAD1 and a non physiological cytoprotective effect for overexpressed TEAD1. Moreover we show that the apoptotic resistance conferred by altered TEAD1 expression is mediated by the transcriptional up-regulation of Livin, a member of the Inhibitor of Apoptosis Protein (IAP) family. In addition, we show that overexpression of a repressive form of TEAD1 can induce Livin up-regulation, indicating that the effect of TEAD1 on Livin expression is indirect and favoring a model in which TEAD1 activates a repressor of Livin by interacting with a limiting cofactor that gets titrated upon TEAD1 up-regulation. Interestingly, we show that overexpression of a mutated form of TEAD1 (Y421H) implicated in Sveinsson's chorioretinal atrophy that strongly reduces its interaction with YAP as well as its activation, can induce Livin expression and protect cells from induced apoptosis, suggesting that YAP is not the cofactor involved in this process. CONCLUSIONS/SIGNIFICANCE: Taken together our data reveal a new, Livin-dependent, apoptotic role for TEAD1 in mammals and provide mechanistic insight downstream of TEAD1 deregulation in cancers.

Integration of differentiation signals during indirect flight muscle formation by a novel enhancer of Drosophila vestigial gene.

The gene vestigial (vg) plays a key role in indirect flight muscle (IFM) development. We show here that vg is controlled by the Notch anti-myogenic signaling pathway in myoblasts and is regulated by a novel 822 bp enhancer during IFM differentiation. Interestingly, this muscle enhancer is activated in developing fibers and in a small number of myoblasts before the fusion of myoblasts with the developing muscle fibers. Moreover, we show that this enhancer is activated by Drosophila Myocyte enhancing factor 2 (MEF2), Scalloped (SD) and VG but repressed by Twist, demonstrating a sensitivity to differentiation in vivo. In vitro experiments reveal that SD can directly bind this enhancer and MEF2 can physically interact with both SD and TWI. Cumulatively, our data reveal the interplay between different myogenic factors responsible for the expression of an enhancer activated during muscle differentiation.

SCALLOPED interacts with YORKIE, the nuclear effector of the hippo tumor-suppressor pathway in Drosophila.

In Drosophila, SCALLOPED (SD) belongs to a family of evolutionarily conserved proteins characterized by the presence of a TEA/ATTS DNA-binding domain [1, 2]. SD physically interacts with the product of the vestigial (vg) gene, where the dimer functions as a master gene controlling wing formation [3, 4]. The VG-SD dimer activates the transcription of several specific wing genes, including sd and vg themselves [5, 6]. The dimer drives cell-cycle progression by inducing expression of the dE2F1 transcription factor [7], which regulates genes involved in DNA replication and cell-cycle progression. Recently, YORKIE (YKI) was identified as a transcriptional coactivator that is the downstream effector of the Hippo signaling pathway, which controls cell proliferation and apoptosis in Drosophila[8]. We identified SD as a partner for YKI. We show that interaction between YKI and SD increases SD transcriptional activity both ex vivo in Drosophila S2 cells and in vivo in Drosophila wing discs and promotes YKI nuclear localization. We also show that YKI overexpression induces vg and dE2F1 expression and that proliferation induced by YKI or by a dominant-negative form of FAT in wing disc is significantly reduced in a sd hypomorphic mutant context. Contrary to YKI, SD is not required in all imaginal tissues. This indicates that YKI-SD interaction acts in a tissue-specific fashion and that other YKI partners must exist.

Cell cycle genes regulate vestigial and scalloped to ensure normal proliferation in the wing disc of Drosophila melanogaster.

In Drosophila, the Vestigial-Scalloped (VG-SD) dimeric transcription factor is required for wing cell identity and proliferation. Previous results have shown that VG-SD controls expression of the cell cycle positive regulator dE2F1 during wing development. Since wing disc growth is a homeostatic process, we investigated the possibility that genes involved in cell cycle progression regulate vg and sd expression in feedback loops. We focused our experiments on two major regulators of cell cycle progression: dE2F1 and the antagonist dacapo (dap). Our results reinforce the idea that VG/SD stoichiometry is critical for correct development and that an excess in SD over VG disrupts wing growth. We reveal that transcriptional activity of VG-SD and the VG/SD ratio are both modulated by down-expression of cell cycle genes. We also detected a dap-induced sd up-regulation that disrupts wing growth. Moreover, we observed a rescue of a vg hypomorphic mutant phenotype by dE2F1 that is concomitant with vg and sd induction. This regulation of the VG-SD activity by dE2F1 is dependent on the vg genetic background. Our results support the hypothesis that cell cycle genes fine-tune wing growth and cell proliferation, in part, through control of the VG/SD stoichiometry and activity. This points to a homeostatic feedback regulation between proliferation regulators and the VG-SD wing selector.

In vivo analysis of Drosophila deoxyribonucleoside kinase function in cell cycle, cell survival and anti-cancer drugs resistance.

In vitro studies have shown that Drosophila melanogaster has a highly efficient single deoxyribonucleoside kinase (dNK) multisubstrate enzyme. dNK is related to the mammalian Thymidine Kinase 2 (TK2) group involved in the nucleotide synthesis salvage pathway. To study the dNK function in vivo, we constructed transgenic Drosophila strains and impaired the nucleotide de novo synthesis pathway, using antifolates such as aminopterin. Our results show that dNK overexpression rescues both cell death and cell cycle arrest triggered by this anti-cancer drug, and confers global resistance on the fly. Moreover, we show that fly viability and growth depend on the exquisite ratio between dNK expression and its substrate thymidine (dT) in the medium, and that increased dT concentrations trigger apoptosis and a decrease in body mass when dNK is mis-expressed. Finally, dNK expression, unlike that of TK2, is cell cycle dependent and under the control of CyclinE and the dE2F1 transcription factor involved in the G1/S transition. dNK is therefore functionally more closely related to mammalian TK1 than to TK2. This strongly suggests that dNK plays a role in cell proliferation in physiological conditions.

Drosophila Lipid Storage Droplet 2 gene (Lsd-2) is expressed and controls lipid storage in wing imaginal discs.

Lipid droplets are the major neutral lipid storage organelles in higher eukaryotes. The PAT domain proteins (Perilipin, ADRP [adipose differentiation related protein], and TIP47 [tail-interacting 47-kDa protein]) are associated with these structures. Perilipin and ADRP are involved in the regulation of lipid storage and metabolism in mammals. Two genes encoding PAT proteins, Drosophila Lipid Storage Droplet 2 Gene (Lsd-2) and Lsd-2, have been identified in Drosophila. Lsd-2 is expressed in fat bodies and in the female germ line and is involved in lipid storage in these tissues. We showed that Lsd-2 is expressed in third-instar wing imaginal discs in Drosophila, with higher levels in the wing pouch, which corresponds to the presumptive wing region of the wing disc. This specific expression pattern is correlated with a high level of neutral lipid accumulation. We also showed that neutral lipid deposition in the wing disc is severely reduced in an Lsd-2 mutant and is increased with Lsd-2 overexpression. Finally, we showed that overexpression of the vestigial (vg) pro-wing gene induces Lsd-2 expression, suggesting that Lsd-2 mediates a vg role during wing formation. Our results suggest that Lsd-2 function is not restricted to tissues directly involved in lipid storage and could play additional roles during development.

Interaction between apterous and early expression of vestigial in formation of the dorso-ventral compartments in the Drosophila wing disc.

BACKGROUND: Compartment formation is a developmental process that requires the existence of barriers against intermixing between cell groups. In the Drosophila wing disc, the dorso-ventral (D/V) compartment boundary is defined by the expression of the apterous (ap) selector gene in the dorsal compartment. AP activity is under control of dLMO which destabilizes the formation of the AP-CHIP complex. RESULTS: We report that D/V boundary formation in the wing disc also depends on early expression of vestigial (vg). Our data suggest that vg is already required for wing cell proliferation before D/V compartmentalization. In addition, we show that over-expression of vg can, to some extent, rescue the effect of the absence of ap on D/V boundary formation. Early VG product regulates AP activity by inducing dLMO and thus indirectly regulating ap target genes such as fringe and the PSalpha1 and PSalpha2 integrins. CONCLUSION: Normal cell proliferation is necessary for ap expression at the level of the D/V boundary. This would be mediated by vg, which interacts in a dose-dependent way with ap.

Suppressors of the vestigial mutant phenotype increase the level of expression of the gene.

Suppressor genes of the vestigial phenotype have been isolated in a wild-type population. These suppressors have an effect on different wing mutants and are allele-specific in the case of vestigial. In a vgBG background they produced overgrowth of the imaginal wing disc. They also induce cell death in the wild-type strain and alter the distribution of cell death in the mutant strain. Expression of vestigial is increased in the wing disc only. Hypotheses formed to determine the nature of these suppressors are in favor of a direct interaction between these genes and vestigial.